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Coordination of chromosome replication, segregation and cell division in Caulobacter crescentus Jensen, Rasmus Bugge Publication date: 2006 Document Version Publisher's PDF, also known as Version of record Citation for published version (APA): Jensen, R. B. (2006). Coordination of chromosome replication, segregation and cell division in Caulobacter crescentus. Poster session presented at EMBO Workshop on "Cell Cycle and Cytoskeletal Elements in Bacteria", København, Denmark. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the public portal. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 02. maj. 2017 Coordination of chromosome replication, segregation and cell division in Caulobacter crescentus Rasmus B. Jensen Department of Life Sciences and Chemistry, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark. Abstract Identification of the dif site In Caulobacter crescentus, progression through the cell cycle is coupled to a cellular differentiation program. Here, the terminus region of the Caulobacter chromosome were identified and the temporal coordination between multiple different cell cycle events was characterized. A very short delay between initiation of DNA replication and origin movement is observed, indicating absence of cohesion between newly replicated chromosomes. However, completely replicated terminus regions stay associated until shortly before cell division. Cell constriction take place over a non-separated nucleoid, indicating absence of nucleoid occlusion of cell division in Caulobacter. A putative dif recombination site near the position where the GGGCRGGG motifs switch strands was identified. To verify that this site is the Caulobacter dif site, strains with deletions of the putative dif site, xerC and xerD were constructed. Δdif ΔxerD Introduction Caulobacter crescentus exists as morphologically and functionally distinct cell types and cellular differentiation is an integral part of its cell cycle (1). Asymmetric cell division gives two different progeny cells: A non-motile stalked cell with a cylindrical extension (a stalk) at one pole and a motile swarmer cell that possesses a single polar flagellum. The stalked cell initiates chromosome replication and cell elongation immediately after cell division, whereas the swarmer cell is unable to do so. After a defined period, the swarmer cell differentiates into a stalked cell by shedding the flagellum and synthesizing a stalk at the same pole. Chromosome replication is initiated during the swarmer-to-stalked cell transition. In the middle of the cell cycle, a new flagellum is synthesized at the pole opposite the stalk and cell division is initiated. Since the same chromosome segregation and cell division defect are observed in 2 - 4 % of the cells for all three strains, the site is the Caulobacter dif site. The site is unusual since it is located close to genes with functions in central metabolism. The XerC binding site in Caulobacter dif diverges very significantly from other known sites. Terminus movement and separation Coordination between cell cycle progression, terminus movement and separation was examined using a strain with the terminus region of the chromosome tagged with CFP-LacI (2). 1 min 20 min 40 min 60 min 80 min 100 min Phase contrast Terminus Membrane Terminus Origin movement 1 min 20 min 40 min 60 min 80 min 100 min Phase contrast Origin Membrane Origin 100% 80% DNA replication initiated 60% % of chromosome replicated 40% DNA replication completed 80% % of cells % of cells 100% The swarmer cell possesses a single copy of the chromosome, which is oriented with the origin-proximal region located close to the flagellated pole of the cell. After initiation of DNA replication, one of the newly replicated origins moves to the opposite pole of the cell. To characterize the coordination between DNA replication and chromosome movement, a strain where the origin-proximal region of the chromosome is tagged with CFP-LacI (2) was synchronized and the cells at the different stages of the cell cycle were characterized by fluorescence microscopy and flow cytometry. Cells divided 20% Smooth, 1 terminus focus 60% Indented, 1 terminus focus 40% Indented, 2 terminus foci 20% 0% 0% 0 20 40 60 80 100 0 20 Time [min] 40 60 80 100 Time [min] The terminus-proximal part of the chromosome gradually moves from the pole opposite the stalk to mid-cell during the DNA replication process. The completely replicated terminus regions stay associated with each other for an extended period of time after chromosome replication is completed. The terminus regions disassociate shortly before the dividing cells separate. Chromosome trapping by the septum 100% DNA replication initiated % o f c e lls 80% 1 origin focus 60% 2 origin foci 40% 20% Cells divided Membrane staining show cytoplasmic membrane invagination before separation of the replicated terminus foci (see above). To examine coordination between cell division and nucleoid separation, the chromosomal DNA in synchronized cells were stained using DAPI. Chromosomal DNA is present in the entire cell at all stages during the cell cycle, no DNA-free regions are observed. 0% 0 20 40 60 80 1 min 100 20 min 40 min 60 min 80 min 100 min Time [min] A very short delay between initiation of DNA replication and origin movement is observed, indicating either absence of or only a very brief period of cohesion between the newly replicated origin-proximal parts of the Caulobacter chromosome. Chromosome Identification of the terminus region To identify the Caulobacter terminus region, a combined bioinformatics and experimental approach was used. Most bacterial genomes contains skewed sequences that abruptly switch strands near the origin and terminus. Two highly significantly skewed sequences were identified, that abrubtly switch strand near postion 1.95 Mb opposite the origin on the genetic map. GCGGTGGT Phase contrast Phase contrast Chromosome GGGCRGGG Invagination of the cytoplasmic membrane is observed before the terminus regions separate and two nucleoids are formed, resulting in trapping of a chromosome on either side of the cell division septum. Thus, no nucleoid occlusion of cell division is observed in Caulobacter. Cytoplasmic membrane invagination takes place approximately 20 minutes before separation of the progeny cells, during the final step of the cell division process. The uncoupling between invagination of the cytoplasmic membrane and the outer part of the cell wall could be responsible for the previously observed compartmentalization of the predivisional cell. References 1 Skerker, J. M. & Laub, M. T. (2004) Nat Rev Microbiol 2, 325-337. 2 Viollier, P. H., Thanbichler, M., McGrath, P. T., West, L., Meewan, M., et al. (2004) Proc Natl Acad Sci U S A 101, 92579262. The GGGCRGGG motif is very similar to the DNA motifs from E. coli that control FtsKmediated movement of the terminus-proximal part of the chromosome. Acknowledgements This work was supported by grants from the Danish Natural Sciences Research Council and The Carlsberg Foundation